基于浸泡法的牙鲆耳石锶标记技术研究
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摘要
为了探究耳石微化学标记技术在牙鲆增殖放流中的应用,以全长(1.56±0.16)cm的牙鲆幼鱼为研究对象,在其养殖水中分别添加0.5 mg/L、2 mg/L、8 mg/L、32 mg/L SrCl_2·6H_2O,以无添加SrCl_2·6H_2O组为对照组,人为改变牙鲆幼鱼生存环境中Sr~(2+)浓度72 h,研究不同浓度SrCl_2·6H_2O对牙鲆耳石锶标记效果。标记期间每24 h换水1次,换水量为50%,换水后补加SrCl_2·6H_2O至原始浓度,记录各浓度组标记期间及标记后的鱼苗死亡数量。利用X射线电子探针微区分析仪(EPMA)对耳石样本进行分析,定量线分析结果表明, 0.5 mg/L、2 mg/L SrCl_2·6H_2O浸染不能形成牙鲆耳石锶标记。8 mg/L浓度组出现Sr峰区, Sr/Ca (Sr:Ca×1000)均值为4.50～6.60, Sr峰值为5.49～8.49。32 mg/L浓度组Sr峰区均值为4.83～7.55, Sr峰值为5.62～11.04。这说明8 mg/L、32 mg/L SrCl_2·6H_2O可对牙鲆耳石进行锶标记。但8 mg/L浓度组出现未标记样本,且Sr峰区均值较32 mg/L浓度组低。32 mg/L浓度组Sr/Ca比值显著增高,即锶标记的Sr/Ca比值为6.61±0.86,与标记前和对照组Sr/Ca比值差异均显著(P<0.05),锶标记率100%。面分布分析(mapping analysis)结果表明,从8 mg/L SrCl_2·6H_2O浓度组开始,耳石出现红色"高锶标记环",与耳石本底颜色对比明显,且随浓度升高,红色"高锶标记环"由浅变深,锶标记效果明显增强。单因素方差分析和Duncan多重比较结果显示,各浓度组全长、体重与对照组差异不显著(P>0.05),不同浓度的SrCl_2·6H_2O对牙鲆幼鱼生长无影响。经χ2检验,各浓度组在浸泡标记期间死亡率及标记后养殖90 d的累积死亡率无显著性差异(P>0.05),不同浓度的SrCl_2·6H_2O对牙鲆幼鱼死亡率无显著影响。因此, 32 mg/L SrCl_2·6H_2O为最佳标记浓度。
To explore the method of microchemistry marking on the otolith of Japanese flounder, Paralichthys olivaceus, during the process of proliferation and release, the juveniles with a total length of(1.56±0.16) cm were immersed at four different concentrations(0.5, 2, 8, and 32 mg/L) of SrCl_2·6H_2O for 72 h to detect the Sr sedimentation effect. During the immersion period, half of the water was exchanged by fresh seawater daily, and SrCl_2·6H_2O were added to maintain constant concentration for each group. The exogenous Sr sedimentation in otoliths was detected by X-ray electron probe microanalyzer(EPMA). Results of line transect analysis showed that the strontium marking on otoliths could not be formed after the immersion of two concentrations(0.5 mg/L and 2 mg/L). The Sr high area appeared in the 8 mg/L concentration group with a 5.49–8.49 peak value, and the mean value of the Sr/Ca(Sr : Ca×1000) ratio was 4.50–6.60. Furthermore, in the 32 mg/L group, the mean value of the Sr/Ca ratio was 6.61±0.86, and the peak value of Sr was 5.62–11.04. This means that otoliths of Paralichthys olivaceus can be marked with strontium by the method of SrCl_2·6H_2O immersion. However, in the 8 mg/L concentration group there were also unlabeled samples. and the mean value of Sr/Ca was lower than that of the 32 mg/L group. In the 32 mg/L group, the strontium marking rate was 100%, and the mean value of the Sr/Ca ratio was 6.61±0.86, which was significantly different(P<0.05) compared with that of the control group. The EPMA mapping analysis showed that the red "high strontium marking ring" in the otolith appeared in the 8 mg/L group and 32 mg/L group, and the color of the "high strontium marking ring" was deepened with increasing of concentration. One-way analysis results showed that the total length and weight was not significantly different between the experimental groups and the control group(P>0.05), which means that the SrCl_2·6H_2O immersion did not affect the growth of young fish. A Chi-square test(P>0.05) showed there were no significant differences in mortality rate among the different concentration groups. Overall, 32 mg/L SrCl_2·6H_2O was the best marker concentration.
To explore the method of microchemistry marking on the otolith of Japanese flounder, Paralichthys olivaceus, during the process of proliferation and release, the juveniles with a total length of(1.56±0.16) cm were immersed at four different concentrations(0.5, 2, 8, and 32 mg/L) of SrCl_2·6H_2O for 72 h to detect the Sr sedimentation effect. During the immersion period, half of the water was exchanged by fresh seawater daily, and SrCl_2·6H_2O were added to maintain constant concentration for each group. The exogenous Sr sedimentation in otoliths was detected by X-ray electron probe microanalyzer(EPMA). Results of line transect analysis showed that the strontium marking on otoliths could not be formed after the immersion of two concentrations(0.5 mg/L and 2 mg/L). The Sr high area appeared in the 8 mg/L concentration group with a 5.49–8.49 peak value, and the mean value of the Sr/Ca(Sr : Ca×1000) ratio was 4.50–6.60. Furthermore, in the 32 mg/L group, the mean value of the Sr/Ca ratio was 6.61±0.86, and the peak value of Sr was 5.62–11.04. This means that otoliths of Paralichthys olivaceus can be marked with strontium by the method of SrCl_2·6H_2O immersion. However, in the 8 mg/L concentration group there were also unlabeled samples. and the mean value of Sr/Ca was lower than that of the 32 mg/L group. In the 32 mg/L group, the strontium marking rate was 100%, and the mean value of the Sr/Ca ratio was 6.61±0.86, which was significantly different(P<0.05) compared with that of the control group. The EPMA mapping analysis showed that the red "high strontium marking ring" in the otolith appeared in the 8 mg/L group and 32 mg/L group, and the color of the "high strontium marking ring" was deepened with increasing of concentration. One-way analysis results showed that the total length and weight was not significantly different between the experimental groups and the control group(P>0.05), which means that the SrCl_2·6H_2O immersion did not affect the growth of young fish. A Chi-square test(P>0.05) showed there were no significant differences in mortality rate among the different concentration groups. Overall, 32 mg/L SrCl_2·6H_2O was the best marker concentration.
引文
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[3]Liu Q.Japanese flounder marking techniques and juveniles released for stock enhancement[D].Qingdao:Ocean University of China,2009.[刘奇.褐牙鲆标记技志与增殖放流试验研究[D].青岛:中国海洋大学,2009.]
[4]Liu Z L,Xu Y J,Liu X Z,et al.Tagging of Japanese flounder using T-bar tags[J].Progress in Fishery Sciences,2013,34(6):80-86.[刘芝亮,徐永江,柳学周,等.T型标志牌标记牙鲆苗种[J].渔业科学进展,2013,34(6):80-86.]
[5]Tong A P,Si F,Liu H J,et al.Application of mtDNA and microsatellite markers to distinguish the released or non-released Japanese flounder Paralichthys olivaceus[J].Journal of Fishery Sciences of China,2015,22(4):630-637.[童爱萍,司飞,刘海金,等.mt DNA和微卫星标记在放流牙鲆和非放流牙鲆鉴定中的应用[J].中国水产科学,2015,22(4):630-637.]
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[7]Gao Y H,Li Z,Qiao L,et al.Raman and FTIR characteristics of otolith of ornamental carp[J].Spectroscopy and Spectral Analysis,2009,29(10):2689-2693.[高永华,李卓,乔莉,等.鲤鱼耳石的拉曼及红外光谱特征研究[J].光谱学与光谱分析,2009,29(10):2689-2693.]
[8]Wang Y K,Huang J S,Dai F Q,et al.Insights into population structure of juvenile small yellow croaker(Larimichthys polyactis)in the Yellow Sea and the Bohai Sea from otolith elemental fingerprints[J].Haiyang Xuebao,2016,38(6):32-40.[王玉堃,黄建生,戴芳群,等.黄、渤海小黄鱼耳石元素指纹分析及其在种群补充群体识别中的应用[J].海洋学报,2016,38(6):32-40.]
[9]Xiong Y,Liu H B,Tang J H,et al.Application of otolith microchemistry on reconstruction of migratory patterns and stock discrimination in marine fishes[J].Chinese Bulletin of Life Sciences,2015,27(7):953-959.[熊瑛,刘洪波,汤建华,等.耳石微化学在海洋鱼类洄游类型和种群识别研究中的应用[J].生命科学,2015,27(7):953-959.]
[10]Fu Z D,Xie T M,Song Z B.Studies on elemental fingerprints in otolith of fish[J].Chinese Journal of Applied and Environmental Biology,2007,13(2):278-283.[付自东,谢天明,宋昭彬.鱼类耳石元素指纹研究进展[J].应用与环境生物学报,2007,13(2):278-283.]
[11]Dou S Z,Yokouchi K,Yu X,et al.Reconstructing migratory history of fish using otolith strontium:calciums ratios by EPMA:a case study[J].Oceanologia et Limnologia Sinica,2011,42(4):512-520.[窦硕增,横内一樹,于鑫,等.基于EPMA的耳石Sr:Ca比分析及其在鱼类生活履历反演中的应用实例研究[J].海洋与湖沼,2011,42(4):512-520.]
[12]Chen T T,Jiang T,Lu M J,et al.Microchemistry analysis of otoliths of Coilia nasus and Coilia brachygnathus from the Jingjiang section of the Yangtze River[J].Journal of Lake Sciences,2016,28(1):149-155.[陈婷婷,姜涛,卢明杰,等.基于耳石微化学的长江靖江段长颌鲚与短颌鲚生境履历重建[J].胡泊科学,2016,28(1):149-155.]
[13]Li M M,Jiang T,Khumbanyiwa D D,et al.Reconstruction habitat history of Coilia Nasus from the Hexian section of the Yangtze river in Anhui province by otolith microchemistry[J].Acta Hydrobiologica Sinica,2017,41(5):1054-1061.[李孟孟,姜涛,Khumbanyiwa D D,等.基于耳石微化学的长江安徽和县江段刀鲚生境履历重建[J].水生生物学报,2017,41(5):1054-1061.]
[14]Phillis C C,Ostrach D J,Ingram O B,et al.Evaluating otolith Sr/Ca as a tool for reconstructing estuarine habitat use[J].Canadian Journal of Fisheries and Aquatic Sciences,2011,68:360-373.
[15]Wang C,Liu W,Zhan P R,et al.Exogenous Sr2+sedimentation on otolith of chum salmon embryos[J].Chinese Journal of Applied Ecology,2015,26(10):3189-3194.[王臣,刘伟,战培荣,等.外源Sr2+在大麻哈鱼胚胎耳石上的沉积[J].应用生态学报,2015,26(10):3189-3194.]
[16]Li X Q,Cong X R,Shi J H,et al.Feasibility analysis of releasing individuals of Aristichthys nobilis identification based on otolith Sr markers[J].Journal of Lake Sciences,2017,29(4):914-922.[李秀启,丛旭日,师吉华,等.耳石锶标记在识别鳙(Aristichthys nobilis)放流个体的可行性[J].湖泊科学,2017,29(4):914-922.]
[17]Wickstr?m H,Sj?berg N B.Traceability of stocked eels-the Swedish approach[J].Ecology of Freshwater Fish,2014,23:33-39.
[18]Kullmann B,Hempel M,Thiel R.Chemical marking of European glass eels Anguilla anguilla with alizarin red S and in combination with strontium:in situ evaluation of short-term salinity effects on survival and efficient mass-marking[J].Journal of Fish Biology,2018,92:203-213.
[19]Brown R J,Severin K P.Otolith chemistry analyses indicate that water Sr:Ca is the primary factor influencing otolith Sr:Ca for freshwater and diadromous fish but not for marine fish[J].Canadian Journal of Fisheries and Aquatic Sciences,2009,66:1790-1808.
[20]Li M M,Jiang T,Chen T T,et al.Otolith microchemistry of the estuarine tapertail anchovy coilia nasus from the Anqing section of the Yangtz River and its significance for migration ecology[J].Acta Ecologica Sinica,2017,37(8):2788-2795.[李孟孟,姜涛,陈婷婷,等.长江安庆江段刀鲚耳石微化学及洄游生态学意义[J].生态学报,2017,37(8):2788-2795.]
[21]Yang J,Liu H B.Otolith microchemistry of grey mullet Mugil cephalus from Chongming water in the Yangtze River estuary,China[J].Journal of Fishery Sciences of China,2010,17(4):853-858.[杨健,刘洪波.长江口崇明水域鲻鱼耳石元素微化学分析[J].中国水产科学,2010,17(4):853-858.]
[22]Zhang H,Jiang Y Z,Yuan X W,et al.Strontium marking on otolith of Larimichthys crocea[J].Journal of Fishery Sciences of China,2015,22(6):1270-1277.[张辉,姜亚洲,袁兴伟,等.大黄鱼耳石锶标志技术[J].中国水产科学,2015,22(6):1270-1277.]
[23]Zhang Y,Jiang Y Z,Xu K D,et al.Evaluation on effectiveness of marking juvenile Sparus macrocephalus otoliths with strontium[J].海洋渔业,2018,40(2):171-178.[张翼,姜亚洲,徐开达,等.锶元素对黑鲷幼鱼耳石的标记效果分析[J].Marine Fisheries,2018,40(2):171-178.]
[24]Schroder S L,Knudsen C M,Volk E C.Marking salmon fry with strontium chloride solutions[J].Canadian Journal of Fisheries and Aquatic Sciences,1995,52(6):1141-1149.
[25]Milton D A,Chenery S R.Sources and uptake of trace metals in otoliths of juvenile barramundi(Lates calcarifer)[J].Journal of Experimental Marine Biology and Ecology,2001,264:47-65.
[26]Cabrera W E,Schrooten I,De Broe M E,et al.Strontium and bone[J].Journal of Bone and Mineral Research,1999,14(5):661-668.
[27]Song H J,Liu W,Wang J L,et al.Effects of strontium ion on growth and ATPase activity in muscle of chum salmon(Oncorhynchus keta)juveniles[J].Chinese Journal of Fisheries,2013,26(4):23-28.[宋洪建,刘伟,王继隆,等.锶对大麻哈鱼稚鱼生长发育及肌肉ATP酶活性的影响[J].水产学杂志,2013,26(4):23-28.]
[2]Lei J L.Marine Fish Culture Theory and Techniques[M].Beijing:China Agriculture Press,2005:482.[雷霁霖.海水鱼类养殖理论与技术[M].北京:中国农业出版社,2005:482.]
[3]Liu Q.Japanese flounder marking techniques and juveniles released for stock enhancement[D].Qingdao:Ocean University of China,2009.[刘奇.褐牙鲆标记技志与增殖放流试验研究[D].青岛:中国海洋大学,2009.]
[4]Liu Z L,Xu Y J,Liu X Z,et al.Tagging of Japanese flounder using T-bar tags[J].Progress in Fishery Sciences,2013,34(6):80-86.[刘芝亮,徐永江,柳学周,等.T型标志牌标记牙鲆苗种[J].渔业科学进展,2013,34(6):80-86.]
[5]Tong A P,Si F,Liu H J,et al.Application of mtDNA and microsatellite markers to distinguish the released or non-released Japanese flounder Paralichthys olivaceus[J].Journal of Fishery Sciences of China,2015,22(4):630-637.[童爱萍,司飞,刘海金,等.mt DNA和微卫星标记在放流牙鲆和非放流牙鲆鉴定中的应用[J].中国水产科学,2015,22(4):630-637.]
[6]Xu Y J,Liu X Z,Zhang K,et al.Tagging juvenile Japanese flounder(Paralichthys olivaceus)with coded wire tags[J].Progress in Fishery Sciences,2017,38(1):168-174.[徐永江,柳学周,张凯,等.编码金属标签对牙鲆苗种标记的效果[J].渔业科学进展,2017,38(1):168-174.]
[7]Gao Y H,Li Z,Qiao L,et al.Raman and FTIR characteristics of otolith of ornamental carp[J].Spectroscopy and Spectral Analysis,2009,29(10):2689-2693.[高永华,李卓,乔莉,等.鲤鱼耳石的拉曼及红外光谱特征研究[J].光谱学与光谱分析,2009,29(10):2689-2693.]
[8]Wang Y K,Huang J S,Dai F Q,et al.Insights into population structure of juvenile small yellow croaker(Larimichthys polyactis)in the Yellow Sea and the Bohai Sea from otolith elemental fingerprints[J].Haiyang Xuebao,2016,38(6):32-40.[王玉堃,黄建生,戴芳群,等.黄、渤海小黄鱼耳石元素指纹分析及其在种群补充群体识别中的应用[J].海洋学报,2016,38(6):32-40.]
[9]Xiong Y,Liu H B,Tang J H,et al.Application of otolith microchemistry on reconstruction of migratory patterns and stock discrimination in marine fishes[J].Chinese Bulletin of Life Sciences,2015,27(7):953-959.[熊瑛,刘洪波,汤建华,等.耳石微化学在海洋鱼类洄游类型和种群识别研究中的应用[J].生命科学,2015,27(7):953-959.]
[10]Fu Z D,Xie T M,Song Z B.Studies on elemental fingerprints in otolith of fish[J].Chinese Journal of Applied and Environmental Biology,2007,13(2):278-283.[付自东,谢天明,宋昭彬.鱼类耳石元素指纹研究进展[J].应用与环境生物学报,2007,13(2):278-283.]
[11]Dou S Z,Yokouchi K,Yu X,et al.Reconstructing migratory history of fish using otolith strontium:calciums ratios by EPMA:a case study[J].Oceanologia et Limnologia Sinica,2011,42(4):512-520.[窦硕增,横内一樹,于鑫,等.基于EPMA的耳石Sr:Ca比分析及其在鱼类生活履历反演中的应用实例研究[J].海洋与湖沼,2011,42(4):512-520.]
[12]Chen T T,Jiang T,Lu M J,et al.Microchemistry analysis of otoliths of Coilia nasus and Coilia brachygnathus from the Jingjiang section of the Yangtze River[J].Journal of Lake Sciences,2016,28(1):149-155.[陈婷婷,姜涛,卢明杰,等.基于耳石微化学的长江靖江段长颌鲚与短颌鲚生境履历重建[J].胡泊科学,2016,28(1):149-155.]
[13]Li M M,Jiang T,Khumbanyiwa D D,et al.Reconstruction habitat history of Coilia Nasus from the Hexian section of the Yangtze river in Anhui province by otolith microchemistry[J].Acta Hydrobiologica Sinica,2017,41(5):1054-1061.[李孟孟,姜涛,Khumbanyiwa D D,等.基于耳石微化学的长江安徽和县江段刀鲚生境履历重建[J].水生生物学报,2017,41(5):1054-1061.]
[14]Phillis C C,Ostrach D J,Ingram O B,et al.Evaluating otolith Sr/Ca as a tool for reconstructing estuarine habitat use[J].Canadian Journal of Fisheries and Aquatic Sciences,2011,68:360-373.
[15]Wang C,Liu W,Zhan P R,et al.Exogenous Sr2+sedimentation on otolith of chum salmon embryos[J].Chinese Journal of Applied Ecology,2015,26(10):3189-3194.[王臣,刘伟,战培荣,等.外源Sr2+在大麻哈鱼胚胎耳石上的沉积[J].应用生态学报,2015,26(10):3189-3194.]
[16]Li X Q,Cong X R,Shi J H,et al.Feasibility analysis of releasing individuals of Aristichthys nobilis identification based on otolith Sr markers[J].Journal of Lake Sciences,2017,29(4):914-922.[李秀启,丛旭日,师吉华,等.耳石锶标记在识别鳙(Aristichthys nobilis)放流个体的可行性[J].湖泊科学,2017,29(4):914-922.]
[17]Wickstr?m H,Sj?berg N B.Traceability of stocked eels-the Swedish approach[J].Ecology of Freshwater Fish,2014,23:33-39.
[18]Kullmann B,Hempel M,Thiel R.Chemical marking of European glass eels Anguilla anguilla with alizarin red S and in combination with strontium:in situ evaluation of short-term salinity effects on survival and efficient mass-marking[J].Journal of Fish Biology,2018,92:203-213.
[19]Brown R J,Severin K P.Otolith chemistry analyses indicate that water Sr:Ca is the primary factor influencing otolith Sr:Ca for freshwater and diadromous fish but not for marine fish[J].Canadian Journal of Fisheries and Aquatic Sciences,2009,66:1790-1808.
[20]Li M M,Jiang T,Chen T T,et al.Otolith microchemistry of the estuarine tapertail anchovy coilia nasus from the Anqing section of the Yangtz River and its significance for migration ecology[J].Acta Ecologica Sinica,2017,37(8):2788-2795.[李孟孟,姜涛,陈婷婷,等.长江安庆江段刀鲚耳石微化学及洄游生态学意义[J].生态学报,2017,37(8):2788-2795.]
[21]Yang J,Liu H B.Otolith microchemistry of grey mullet Mugil cephalus from Chongming water in the Yangtze River estuary,China[J].Journal of Fishery Sciences of China,2010,17(4):853-858.[杨健,刘洪波.长江口崇明水域鲻鱼耳石元素微化学分析[J].中国水产科学,2010,17(4):853-858.]
[22]Zhang H,Jiang Y Z,Yuan X W,et al.Strontium marking on otolith of Larimichthys crocea[J].Journal of Fishery Sciences of China,2015,22(6):1270-1277.[张辉,姜亚洲,袁兴伟,等.大黄鱼耳石锶标志技术[J].中国水产科学,2015,22(6):1270-1277.]
[23]Zhang Y,Jiang Y Z,Xu K D,et al.Evaluation on effectiveness of marking juvenile Sparus macrocephalus otoliths with strontium[J].海洋渔业,2018,40(2):171-178.[张翼,姜亚洲,徐开达,等.锶元素对黑鲷幼鱼耳石的标记效果分析[J].Marine Fisheries,2018,40(2):171-178.]
[24]Schroder S L,Knudsen C M,Volk E C.Marking salmon fry with strontium chloride solutions[J].Canadian Journal of Fisheries and Aquatic Sciences,1995,52(6):1141-1149.
[25]Milton D A,Chenery S R.Sources and uptake of trace metals in otoliths of juvenile barramundi(Lates calcarifer)[J].Journal of Experimental Marine Biology and Ecology,2001,264:47-65.
[26]Cabrera W E,Schrooten I,De Broe M E,et al.Strontium and bone[J].Journal of Bone and Mineral Research,1999,14(5):661-668.
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